| In the nature,sulfur element occupies a very important position,it is the tenth element in the universe,and the fifth on the earth.Sulfur is found in biological molecules such as enzymes,proteins,sugars,nucleic acids,vitamin cofactors and metabolites.Sulfur has extensive valency,and its chemical property is active.Sulfur components with valence from-2 to+6 are common in our lives,for example,hydrogen sulfide is widely used in mammalian cells as a signaling molecule involved in the regulation of the nervous system and vasodilation,Sulfane sulfur has antioxidant properties and is involved in cell regulation,sulfur elementals and sulfites act as electron acceptors or donors to participate in redox reactions in microorganisms,sulfates can assimilate in the gut to produce cysteine and methionine.Many researchers have paid attention to the exploration of sulfur metabolism pathway in microorganisms,such as the study of acidophilic bacteria,archaea and some autotrophic bacteria.Heterotrophic bacteria have large number and diversity,so the sulfur metabolism study of heterotrophic microorganisms is also important.Our laboratory are focusing on the sulfide metabolism process of heterotrophic microorganisms,we hope to provide a theoretical basis for the treatment of environmental sulfur pollution by studing the sulfides redox mechanism of heterotrophic microorganisms.We identified a hydrogen sulfide(H2S)oxidation pathway in the heterotrophic Cupriavidus pinatubonesis JMP134,a sulfide:quinone oxidoreductase(SQR)-persulfide dioxygenase(PDO)system that oxidizes H2S to thiosulfate.The main reaction process is that SQR first oxidizes H2S to polysulfides,and the polysulfides react with intracellular GSH to generate GSSH under the action of sulfur transferase(ST).GSSH,as the substrate of PDO,continues to be oxidized to sulfite.Sulfite then spontaneously reacts with polysulfides to generate thiosulfate.The thiosulfate can be further oxidized to sulfate in bacteria that contain the SOX system.In this metabolic system,polysulfides are important intermediate.As SQR is a peripheral protein toward the cytoplast in C.pinatubonesis JMP134,so the polysulfides generated by SQR catalyzing H2S can be directly utilized by intracellular ST and PDO.However,some studies have shown that SQR and PDO may not exist simultaneously in some bacteria.Some bacteria only have PDO,but can also use the polysulfides from the external environment,how the polysulfides from the external environment entering into the bacterial cells has been less studied.Since polysulfides can be interconverted with H2S in the environment,excessive polysulfides in the environment will generate excessive H2S in the air,resulting in environmental pollution,so it is necessary to study the transport of polysulfides in microorganis and usage of microorganisms to remove the excessive polysulfides from the environments.Literatures showed that the PmpAB proteins in the Serratia sp.strain ATCC 39006 was predicted to transport sulfur compounds,by searching similar PmpAB proteins using Blastp,we found that they belong to the YedE proteins family.There are a wide variety of strains containing YedE proteins,but there are few reports on the function of YedE proteins.CpYedE1 and CpYedE2 from C.pinatubonesis show similar sequence to PmpAB,thus we initiated the study of YedEl-YedE2 proteins can transport polysulfides into the cell.YedEl and YedE2 were assumed to be membrane proteins.By the prediction of TMHMM 2.0 software,each YedE has 4 transmembrane helices.The C and N terminals of CpYedEl protein were both periplasmic located whiie the C and N terminals of CpYedE2 protein were both inside the cytoplasm.We first overexpressed CpYedE1 and CpYedE2 proteins in E.coli BL21(DE3)and detected the membrane localization of CpYedEl and CpYedE2 by Western-blot and fluorescent fusion protein techniques.Then we added polysulfide exogenously,and detected the changes of extracellular and intracellular polysulfides in YedEl-YedE2 overexpressing strains by RSS,PsGFP and HPLC methods,hoping to demonstrate that YedEl-YedE2 can promote poly sulfide transport.For proving the function and transport direction of YedEl-YedE2 further,We overexpressed YedEl-YedE2 in strains containing PDO protein and found that strains containing YedEl-YedE2 protein had a faster consumption rate of polysulfides by whole cell analysis experiment,which proved that YedEl-YedE2 could accelerate the transport of polysulfides into the cell twice.The function of YedEl-YedE2 to transport polysulfides into the cell was again demonstrated by cell rupture experiment.At the same time,the transport direction of YedEl-YedE2 was tested,and it was found that YedEl-YedE2 could only promote the transport of polysulfides from extracellular to intracellular.By detecting the accumulation and production of substrate,it was proved that it could accelerate the transport of polysulfides from periplasmic space to cytoplasm.By adding different substrates exogenously,it was proved that YedEl-YedE2 could transport polysulfides but not GSSH.Finally,whether there was the binding effect between YedEl-YedE2 and polysulfides was detected through MST experiment,and the result illustrated We didn’t detect the binding effect between YedEl-YedE2 and polysulfides. |
